Occurrence of Aflatoxins, Fumonisin B1, and Zearalenone in Foods and Feeds in Botswana

1670

BUPE A. SIAME,* SISAl F. MPUCHANE, BERHANU A. GASHE, JOSEPH ALLOTEY, AND GETACHEW 1EFFERA

Department of Biological Sciences, University of Botswana, Private Bag 0022, Gaborone, Botswana

MS 98-116: Received 29 Aprill998/Accepted 31 July 1998

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Sorghum and maize form the main dietary staple foods in Botswana. Other products such as peanuts, peanut butter, phane (an edible larval stage of an emperor moth 1mbrasia belina Westwood), and pulses (cowpeas and beans) are also widely used as food and for the manufacture of feeds. These important food and feed commodities were analyzed for the presence of afiatoxins, fumonisin B I, and zearalenone. Afiatoxins were detected in 40% of the samples analyzed. The concentration of total afiatoxins ranged from 0.1 to 64 Ilg/kg. The mean concentration ranged from 0.3 Ilg/kg in sorghum to 23 Ilg/kg in peanut butter. Peanut butter samples were the most contaminated (71 %). No afiatoxins were detected in maize. Fumonisin Bl was detected in 36% of the samples. Maize samples were the most contaminated (85% of the samples) with the concentration ranging from 20 to 1,270 Ilg/kg. No fumonisin B I was detected in peanuts, phane, and beans. Zearalenone was only found in 2.6% of the samples analyzed at 40 Ilg/kg. Afiatoxins were the most common toxins detected in foods and feeds in Botswana. However, fumonisin B I was more prevalent in maize than afiatoxins or zearalenone.

Maize, sorghum, and cassava comprise the major com- produce a variety of mycotoxins with widely divergent ponents of the human diet in Africa. Other crops such as biological and toxicological effects in humans and animals wheat, rice, millet, groundnuts, pulses (cowpeas and beans), consuming the contaminated commodities. Surveys carried and sunflower are also used but to a lesser extent. Warm wet out in some European countries such as Italy, France, and conditions, prevalent in many parts of Africa, are ideal for Spain have shown that Fusarium moniliforme is the most fungal proliferation. Poor harvesting, handling, and storage frequently isolated fungal species from maize in the field, practices also contribute to fungal growth and mycotoxin harvested maize, and maize-based commodities (5, 23). In production. The main genera of fungi that produce mycotox- African countries such as Lesotho, Nigeria, and Zimbabwe, ins are Aspergillus, Fusarium, and Penicillium. F. moniliforme is also common on maize, millet, and In Africa as in most developing countries the aflatoxins sorghum (21). are the most important mycotoxins from the point of view of Among the mycotoxins produced by F. moniliforme, the occurrence, toxicity, and economy. The aflatoxins are pro- fumonisins are among the most toxic and have been duced primarily by strains of Aspergillus flavus and Aspergil- associated with problems in human and animal health. Of lus parasiticus. The aflatoxins commonly isolated from the six fumonisins that have been described, fumonisins B 1, foods and feeds are aflatoxins Bl, B2, Gl, and G2. These B2, and B3 are the major ones usually isolated from fungal toxins are primarily a problem in oil nuts such as peanuts cultures on maize and from naturally contaminated maize and sunflower, although cereals such as maize, sorghum, (26). Fumonisin B 1 has been associated with toxicoses of millet, wheat, and rice can also be contaminated (19). animals and is the causal agent of equine leukoencephaloma- Aflatoxins are lethal to experimental animals when lacia (ELEM) and porcine pulmonary edema (PPE) (14, 15). consumed in large doses. Acute toxicity in humans has been Consumption of fumonisin contaminated maize has also reported in Uganda and Kenya (1, 22). Aflatoxins are also been linked to esophageal cancer (6, 11, 24). potent carcinogens and mutagens. Chronic exposure to Zearalenone is also produced by Fusarium species, aflatoxins at sublethal doses has been associated with liver especially Fusarium graminearum. It has been reported to cancer in humans. Although a direct cause-effect relation- contaminate samples from several African countries (8). The ship has not been demonstrated, a general correlation has major effects of zearalenone are estrogenic and mostly affect been established between the incidence of liver cancer in the urogenital system. It produces a condition known as humans in specific areas of Africa and dietary exposure to hyperestrogenism in pigs and has also been implicated in aflatoxins (22). some incidents of precocious puberty changes in children Fusarium species are now also recognized as a major (16). agricultural problem. They occur worldwide on a variety of In Botswana, sorghum and maize form the main dietary plant hosts, and primarily on cereal grains. Fusarium species staple foods. These cereals along with oil nuts such as peanuts and sunflower seeds are widely used for food and * Author for correspondence. Tel: (267) 3552609; Fax: (267) 3552784; animal feed. Mophane worms, the larval stage of the E-mail: [email protected]. emperor moth 1mbrasia belina Westwood, are also an J. Food Prot.,Vol.61, No. 12 AFLATOXINS,FUMONISINBl, AND ZEARALENONEIN FOODS 1671 important protein source for many people in Botswana. The citric acid, and zearalenone was taken up in dichloromethane. The worms (commonly known as phane) are harvested and dichloromethane was evaporated to dryness, and the residue was processed by women and are an important source of taken up in 200 111of chloroform for TLC and HPLC. TLC was livelihood for many of these women. In this study, we report carried out on 0.2-rom silica gel plates (E. Merck, Germany) in diethyl ether-cyc1ohexane (75:25, vol/vol). Quantitation was by the occurrence of aflatoxins (Bl, B2, Gl, and G2), fumoni- visual comparison of the fluorescence intensity with that of sin B 1, and zearalenone in these important food and feed standards. The detection limit was 20 I1g/kg. The identity of commodities. zearalenone was confirmed by HPLC using fluorescence detection (Waters HPLC system). Fluorescence was recorded at "Ex and "Em MATERIALS AND METHODS wavelengths of 280 and 465 nm, respectively. Separation was Sample source and preparation. Maize (and maize meal), achieved using the same column and mobile phase as that for sorghum (and sorghum meal), peanuts, and peanut butter samples, fumonisins. all meant for human consumption, were either collected from storage depots or purchased from retail outlets. The chicken feed Downloaded from http://meridian.allenpress.com/jfp/article-pdf/61/12/1670/1663731/0362-028x-61_12_1670.pdf by guest on 26 September 2021 samples were also purchased from retail outlets. Phane samples RESULTS were purchased from harvesters in the field during the harvest Aflatoxins were detected in 53 of the 134 samples tested seasons and from retailers. All samples were obtained between giving an incidence of 40%. All chicken feed samples January 1996 and December 1997. Sample size varied from 2 to 5 (100%),71 % of the peanut butter, 57% of the phane, 52% of kg except for peanut butter where 500 g bottles were purchased. the peanut, and 16% of the sorghum samples were contami- The dry samples were finely ground in a Buehler laboratory mill and thoroughly mixed before taking aliquots for aflatoxin, fumoni- nated (Table 1). Only maize had no detectable aflatoxins. sin Bl, and zearalenone analysis. Peanut butter samples were Concentration of total aflatoxins (B 1 + B2 + G 1 + G2) in thoroughly mixed before taking a subsample. All analyses were the positive samples ranged from 0.1 flglkg to 64 flg/kg. done in duplicate. Among the aflatoxin positive samples the lowest concentrations were recorded in sorghum (0.1 to 0.5 flg/kg) and Aflatoxin analysis. The toxins were extracted by the BF chicken feed (0.1 to 0.7 flg/kg) samples. High concentrations method (2). Subsamples (50 g) were extracted into methanol-water and partitioned with chloroform. The solvents were evaporated and of aflatoxins were recorded in peanut butter (1.6 to 64 flg/kg) the residue taken tip in 200 111of chloroform for thin-layer and raw peanuts (3.2 to 48 flg/kg). The average concentra- chromatography (TLC) and high-performance liquid chromatogra- tion of total aflatoxins ranged from 0.3 flg/kg in sorghum to phy (HPLC). TLC was carried out on 0.2-mm silica gel plates (E. 23 flg/kg in peanut butter. All the four major aflatoxins (Bl, Merck, Germany). The quantity was determined by visual compari- B2, G 1, and G2) were detected in peanut butter, peanuts, and son of the fluorescence intensity with that of the standards. The phane (Table 2). However, only aflatoxin Bland G 1 were identity and amount of aflatoxins (and their trifluoroacetic acid detected in chicken feed and sorghum samples. derivatives) were confirmed using a Waters HPLC system, Model Fumonisin B 1 was detected in all chicken feed samples 610G pump, and Model 474 scanning fluorescence detector (100%),85% of the maize, and 15% of the sorghum samples (Waters, Milford, Mass.). The fluorescence was recorded at excita- (Table 3). The rest of the samples (peanuts, beans, and tion (t'Ex) and emission (t'Em) wavelengths of 360 and 425 nm, phane) had no detectable fumonisins. The concentration of respectively. Separations were done on anAlphabond C-18 (10 11m) column (300 by 3.9 rom; Sigma, St Louis, Mo.) with a column fumonisin B 1 in the positive samples ranged from 20 to temperature of 30°C. The mobile phase was acetonitrile-water- 1,270 flg/kg. Although the highest concentration was re- acetic acid (45:55:2, vol/vol) at a flow rate of 1 ml/min. The corded in maize (1,270 flg/kg), chicken feed samples had a detection limit was O.ll1g/kg. higher mean concentration (572 flg/kg) than maize (247 flg/kg). The lowest concentration range was recorded in Fumonisin analysis. The fumonisins were analyzed by the sorghum (20 to 60 flg/kg). method given Doko et al. (9). Fumonisins were extracted from 50 g subsamples into methanol-water. The methanol-water extract was Zearalenone was only detected in 2 of the 78 samples passed through a strong anion-exchange cartridge (Supe1co,Belle- analyzed (2.6%) (Table 4). The two samples (maize and fonte, Pa.) and the fumonisins eluted from the cartridge with acetic chicken feed) had zearalenone concentrations of 40 flg/kg acid-methanol. The acetic acid-methanol solvents were evaporated under a gentle stream of nitrogen. Fumonisin B1 was then derivatized with 200 111of o-phthaldialdehyde (OPA). The OPA TABLE 1. Occurrence and relative amounts of total aflatoxins derivatives (10 111)were analyzed by reverse-phase HPLC with (Bl + B2 + Gl + G2) in food and feed commodities in Botswana fluorescence detection (WatersHPLC system). The fluorescence of Occurrence the OPAderivatives was recorded at "Ex and "Em wavelength of 335 (no.positive! % Range, Mean, and 440 nm, respectively. Separations were done on a Hypersil Sample total) contaminated IJg/kg IJg/kg ODS (311m) column (100 by 4.6 rom; Sigma) with a column temperature of 30°C. The mobile phase was methanol-O.l M Peanuts 15/29 52 3.2-48 14 NaHzP04 (75:25, vol/vol) at a flow rate of 1 mlImin. Quantitation Peanut butter 15/21 71 1.6-64 23 was by comparison with reference standards. The detection limit Phane 16/28 57 0.1-10 2.5 was 20 I1g/kg. Chicken feed 4/4 100 0.1-0.7 0.6 Maize (+ meal) 0/33 NDa ND ND Zearalenone analysis. The method by Bennett et al. (4), with Sorghum (+ meal) 3/19 16 0.1-0.5 0.3 some modifications, was used for zearalenone. Zearalenone was Total (all samples) 53/134 40 0.1-64 0.8 extracted from samples (50 g) with chloroform and partitioned with sodium hydroxide. The pH of the aqueous layer was lowered with a ND, not detected. 1672 SIAME ET AL. J. Food Prot., Vol. 61, No. 12

TABLE 2. Distribution of aflatoxin Bl, B2, Gl, and G2 in the variations in aflatoxin contamination of maize in storage. aflatoxin positive samples on sale in Botswana Therefore, there is need for constant surveillance of grains in Occur- the storage silos to determine the levels of aflatoxins. The rence Aflatoxinrange(~g) most contaminated samples were the peanut butter and (no.pos- peanuts. This is consistent with what has been reported by Sample itive!total) Bl B2 Gl G2 others. For example, oil nuts and their products from Nigeria were found to be highly susceptible to contamination by Peanuts 15/29 0.8-16.0 1.6-16.0 1.6-8.0 1.6-16.0 Peanut butter 15/21 3.2-16.0 1.6-20.0 3.2-20.0 1.6-20.0 aflatoxins (3). Phane 16/28 0.1-5.0 0.1-2.0 0.5-3.0 0.2-1.0 The maximum allowable levels of aflatoxms in food Chicken feed 4/4 0.1--D.5 NDa 0.2--D.5 ND intended for human consumption is 20 /-lg/kgor less in many Maize (+ meal) 0/33 ND ND ND ND countries. However, there are only five countries in Africa Sorghum (+meal) 3/19 0.1--D.5 ND 0.2--D.5 ND (South Africa, Zimbabwe, Malawi, Kenya, and Nigeria) Total (all samples) 53/134 ~16.0 ~20.0 ~20.0 ~20.0 with official regulations (25). The peanut butter samples in Downloaded from http://meridian.allenpress.com/jfp/article-pdf/61/12/1670/1663731/0362-028x-61_12_1670.pdf by guest on 26 September 2021 our study had aflatoxin concentrations of up to 64 /-lg/kg. a ND, not detected. Studies in Kenya, Mozambique, and Swaziland showed TABLE 3. Occurrence offumonisins infood and feed commodities positive correlations between aflatoxin levels in the diet and in Botswana the incidence of primary liver cancer. In all these studies the incidence of liver cancer was higher in regions of high Occurrence (no. positive! % Range, Mean, aflatoxin consumption than in regions where aflatoxin Sample total) contaminated Ilglkg Ilglkg consumption was low (13). Considering the high incidence and levels of aflatoxins in peanuts and peanut butter, it is Maize (+ meal) 28/33 85 20-1,270 247 important that regulation of aflatoxins in these food commodi- Sorghum (+ meal) 3/20 15 20-60 43 ties be considered in Botswana. The Food Control Board of Chicken feed 4/4 100 163-1,050 572 Botswana recently set up a committee to draft legislation to Phane 0/20 NDa ND ND Peanuts 0/15 ND ND ND control aflatoxins in foods and feeds. Beans 0/5 ND ND ND Sorghum and maize form the staple diet of many people Total (all samples) 35/97 36 ND-1,270 25 in Botswana. Mpuchane et al. (20) reported the occurrence of Fusarium species on stored maize with F. moniliforme a ND, not detected. being the most prevalent. Fusarium species have also been TABLE 4. Occurrence of zearalenone in selected food and feed isolated from phane samples (12). Fusarium species produce samples on sale in Botswana a variety of mycotoxins that include fumonisins and zearalenone. Occurrence Range, % Since fumonisins are produced by F. moniliforme, high Sample (no. positive! total) contaminated Ilglkg rates of incidence of fumonisins have been reported in maize Maize (+ meal) 1/20 5 40 worldwide (5, 9, 23). Fumonisin B 1 was detected mostly in Sorghum (+ meal) 0/19 0 NDa maize and chicken feed and to a lesser extent in sorghum. In Chicken feed 1/4 25 40 a survey of eastern and southern African cereals and Peanuts 0/15 ND ND cereal-based commodities, fumonisin B 1 was detected in Phane 0/20 ND ND 92 % of the samples analyzed at concentrations ranging from Total (all samples) 2/78 2.6 ND-40 20 to 1,910 /-lg/kg (8). a ND, not detected. FAO (10) has estimated maize intakes of more than 200 g per person per day for people in eastern and southern each. The rest of the samples (sorghum, phane, and peanuts) Africa. On the basis of the average daily intake of 200 g of had no detectable zearalenone. maize per person per day and a mean fumonisin B 1 concentration of 247 /-lg/kgit can be estimated that individu- DISCUSSION als in Botswana are consuming about 50 /-lgof fumomsin B 1 Aflatoxin-producing Aspergillus species are common per day. Fumonisin levels of 10 and 100 /-lg/gof sample are on maize, sorghum, peanuts, and sunflower. In a mycologi- considered dangerous for pigs and horses, respectively (17). cal study of maize and sorghum from grain storage silos in In the Transkei region of South Africa concentrations of up Botswana, up to 41 % of the total fungal counts in maize and to 117,500 /-lg/kg have been reported in maize meant for 25% of those in sorghum were A. flavus and A. parasiticus brewing of beer (24). Consumption of fumonisin-contami- (20). In the same study, total aflatoxin concentrations ranged nated maize in this region has been linked to esophageal from 5 to 50 /-lg/kg in maize and 5 to 25 /-lg/kg in sorghum. cancer (17, 24). Similar associations between consumption Similarly, Aspergillus species have been isolated from phane of maize contaminated with fumonisins and esophageal (12, 20). In this study, aflatoxins were detected in peanuts, cancer have also been reported in China and northeastern peanut butter, phane, chicken feed, and sorghum, but none Italy (6, 11). Although the concentrations reported in this were detected in maize. The absence of aflatoxins in maize study are lower than those reported in samples from (although previous studies have shown some level of Transkei, the fact that fumonisins are prevalent in a staple contamination) suggests that there are seasonal or annual food should be of concern to consumers. No information is J. Food Prot., Vol. 61, No. 12 AFLATOXINS, FUMONISIN Bl, AND ZEARALENONE IN FOODS 1673 available on the cumulative effects of fumonisin B 1 on 8. Doko, M. B., C. Canet, N. Brown, E. W. Sydenham, S. Mpuchane, and human health. To our knowledge only Switzerland has set B. A. Siame. 1996. Natural co-occurrence of fumonisins and zearalenone in cereals and cereal-based foods from eastern and southern official limits (at 1,000 flg/kg) on the levels of fumonisins in Africa. J. Agric. Food Chern. 44:3240-3243. foods (7). 9. Doko, M. B., S. Rapior, S. Visconti, and J. E. Schjoth. 1995. Incidence Zearalenone was only detected in two of the samples and levels of fumonisin contamination in maize genotypes grown in (2.6%) at concentrations of 40 flg/kg each. The low inci- Europe and Africa. J. Agric. Food Chern. 43:429-434. 10. FAO. 1992. Maize in human nutrition. FAO Food and Nutrition Series dence and the low levels found suggest that zearalenone is No. 25. ISBN 95-5-103013-8. FAO, Rome, Italy. not a problem in cereals on sale in Botswana. A similar low 11. Franceschi, S., E. Bidoli, A. E. Baron, and C. La Vecchia. 1990. Maize incidence (12.5%) was found in a survey of cereals and and the risk of cancers of the oral cavity, pharynx, and esophagus in cereal-based commodities from eastern and southern Africa northeastern Italy. J. Nat!. Cancer Inst. 82:1407-1410. 12. Gashe, B. A., S. F. Mpuchane, B. A. Siame, J. Allotey, and G. Teferra. (8). However, high levels of zearalenone have been reported 1997. The microbiology ofphane, an edible caterpillar ofthe emperor in maize samples meant for brewing beer in South Africa (up moth, 1mbrasia belina. J. Food Prot. 60:1376-1380. 13. Groopman, J. D., L. G. Cain, and T. W. Kensler. 1988. Aflatoxin to 10,000 )..lg/kg)(18). 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